Immobilized metal affinity chromatography (IMAC), also known as metal chelate affinity chromatography (MCAC), is a specialized aspect of affinity chromatography. The principle behind IMAC lies in the fact that many transition metal ions, i.e., zinc and copper, can coordinate to the amino acids histidine, cystein, and tryptophan via electron donor groups on the amino acid side chains. To utilize this interaction for chromatographic purposes, the metal ion must be immobilized onto an insoluble support. This can be done by attaching a chelating group to the chromatographic matrix. Most importantly, to be useful, the metal of choice must have a higher affinity for the matrix than for the compounds to be purified.
The most common chelating group used in this technique is iminodiacetic acid (IDA). It is coupled to a matrix such as SEPHAROSE 6B, via a long hydrophilic spacer arm. The spacer arm ensures that the chelating metal is fully accessible to all available binding sites on a protein. Another popular chelating group for IMAC applications is tris(carboxymethyl)-ethylenediamine (TED). This particular group lends different properties to the gel than IDA. TED gels show stronger retention of metal ions and weaker retention of proteins relative to that of IDA gels. TED gels form a complex (single coordination site) vs a chelate (multiple coordination sites for IDA gels. The most commonly used metals for IMAC are zinc and copper; however, nickel cobalt, and calcium have also been used successfully.
The develpment of IMAC in purification processes can be facilated considerably by accurate prediction of the protein affinity of a given protein for IMAC resins before performing separations in the laboratory. If the affinity for an IMAC resin could be reliably and easily predicted from its protein structure, then the reseacher would be better informed when deciding on a development strategy. A protein predicted to have a high affinity, for example could be bound to a resin under relatively stringent conditions and eluted with a simple isocratic step. In contrast, IMAC should not be considered as a primary purification step for a protein predicted to possess a low affinity to the metal-chelating resins.
Furthermore, a protein of only moderate affinity requires a less stringent binding condition and a sophisticated gradient elution. To achieve high resolution and maximum recovery in IMAC, however, knowledge of the relative affinity of proteins to immobilized metals is required. Without prediction of protein-resin affinity, purification development of IMAC may become an unnecessarily time consuming effort which may not yield useful results.
Zn-Chelating Chromatography has been utilized in the clinical production of human interleukin-4 (h IL-4), human interleukin-10 (h IL-10) and human tissue plasminogen activator (h tPA). IMAC relies primarily on the interaction between Histidine (His) and a metal ion reversibly bound to a stationery phase. Although immobilized, Zn is extensively used because its selectivity, other metal ions like Cu.sup.++, Ni.sup.++, and Co.sup.++ are also applied for certain proteins. Interactions between immobilized metals and tryptophan, tyrosine, or cysteine residues of proteins have been reported, however, these are generally weaker interactions. Furthermore, when a histidine lies in close proximity to an aromatic residue or another histidine (e.g. on the same position of successive turns of an alpha helix), a cooperative effect leading to high affinity is observed. Although protein leader sequences ontaining His-Tyr, His-Trp, His-X-X-His, have been engineered to take advantage of this phenomenon, these sequences are relatively rare in nature. With naturally occurring proteins, therefore, one can generalize that affinity of a protein for conventional IMAC resins is dictated by the availability of His side chain, imidazole.
Histidine availability, however, is not simply proportional to the total number of His residues in a protein. This is evident from our experience with IMAC of several recombinant proteins under various chromatographic conditions. Since the protein binding capacity of immobilized metal increases as the buffer pH raises, we subjected these proteins to different binding conditions, varied from pH 6.75 (more stringent condition) to pH 7.5 (less stringent condition). Although both h IL-4 and the soluble domain of murine gamma interferon receptor (m IFN R) each contains 5 His residues per monomer, only h IL-4 can bind quantitatively to Zn-Chelating SEPHAROSE under quite stringent conditions (at pH 7.0). In contrast m .gamma.IFN R does not significantly bind to the immobilized Zn, either at pH 7.0 or pH 7.7, a more favorable binding condition. A similar phenomenon was observed with h IL-10 and h IL-13. There are 3 His residues per monomer in both of these proteins, but only h IL-10 can bind to the immobilized Zn quantitatively a pH 7.5. Additionally, although the soluble domain of h IL-10 receptor (h IL-10 R) and m .gamma.IFN R contain similar numbers of His residues per molecule (7 and 5, respectively), h IL-10 R has remarkably stronger affinity to Zn-Chelating SEPHAROSE. The protein binds quantitatively at pH 6.75, a very stringent condition. This is in contrast to the poor affinity exhibited by m .gamma.IFN R, even under the more favorable binding conditions of pH 7.0 and pH 7.5.
These results clearly show that the total number of His residues does not solely determine affinity to IMAC. Therefore, the ability to predict when a His residue is sufficiently exposed to binding to immobilized metals would provide IMAC development with a valuable tool. Thus there is a need to determine a process by which one can determine whether or not a protein will be a suitable candidate for purification by IMAC and what the optimal conditions are.